The ribosomal hydrolysis of GTP is essential for protein biosynthesis to occur. In eukaryotic cells GTP is hydrolyzed in four distinct steps of this process: initiator aminoacyl-tRNA binding, aminoacyl-tRNA binding during elongation, translocation and termination. From a variety of lines of evidence, it is possible that there is a single GTP hyrolytic site on the ribosome for all of these four steps. The overall purpose of our investigation is to determine wether or not this is true. The research involves purification of reticulocyte EF2 by a novel procedure in which EF2 is covalently bound to NAD-agarose via the diptheria toxin reaction and then this reaction reversed. Two GTP analogues, p-azidophenyl GDP binding sites of reticulocyte EF2 and EF1. The first is a photoaffinity label while the second is a fluorescent probe. The interactions of these compounds with the separate elongation factors and in the presence of ribosomes are compared to gain information on the GTP binding and hydrolysis sites.